REVIEWS

Clinical blockade of PD1 and LAG3 — potential mechanisms of action Linh T. Nguyen and Pamela S. Ohashi

Abstract | Dysfunctional T cells can render the immune system unable to eliminate infections and cancer. Therapeutic targeting of the surface receptors that inhibit T cell function has begun to show remarkable success in clinical trials. In this Review, we discuss the potential mechanisms of action of the clinical agents that target two of these receptors, programmed cell death protein 1 (PD1) and lymphocyte activation gene 3 protein (LAG3). We also suggest correlative studies that may define the predominant mechanisms of action and identify predictive biomarkers.

Exhaustion A state of impaired T cell function that results from chronic exposure to an antigen.

Inhibitory receptors Receptors that negatively regulate cellular function. They may contain immunoreceptor tyrosine-based inhibition (ITIM) motifs. The absence of such receptors or their inhibition by antagonistic antibodies leads to improved cell function.

Immune Therapy Program, Princess Margaret Cancer Centre, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada. e‑mails: [email protected]; [email protected] doi:10.1038/nri3790

T cells have an important role in antiviral and antitumour immune responses. Appropriate activation of antigen-specific T cells leads to their clonal expansion and their acquisition of effector function, and, in the case of cytotoxic T lymphocytes (CTLs), it enables them to specifically lyse target cells. In some situations such as chronic infection, T cell dysfunction occurs as a result of prolonged antigen exposure: the T cell loses the ability to proliferate in the presence of the antigen and progressively fails to produce cytokines and to lyse target cells1. This dysfunctional state has been termed ‘exhaustion’. During chronic viral infection or cancer, dysfunctional T cells have a reduced ability to clear pathogens or to eliminate cancer cells. Many insights have recently been gained into the role of various receptors that negatively regulate T cell function and promote exhaustion. There is now widespread interest in developing therapeutic agents that target these molecules to augment T cell activity. In this Review, we focus on two inhibitory receptors, programmed cell death protein 1 (PD1; also known as CD279) and lymphocyte activation gene 3 protein (LAG3; also known as CD223). We summarize results from clinical trials and discuss potential mechanisms of action. We also suggest correlative studies that may help to define biomarkers of clinical response.

Inhibitory receptors on T cells In 2007, a molecular signature of exhausted T cells was defined from studies of the clone 13 strain of lympho­ cytic choriomeningitis virus (LCMV)2. This virus strain establishes a chronic infection in mice wherein virusspecific T cells persist but lose their effector function3. Gene expression microarray analysis of virus-specific T cells during LCMV clone 13 infection showed upregulation

of a panel of receptors with negative regulatory function compared with T cells from mice infected with the LCMV Armstrong strain, which causes acute infection2. These receptors included cytotoxic T lymphocyte antigen 4 (CTLA4), PD1, LAG3, killer cell lectin-like receptor subfamily G member 1 (KLRG1), T cell immuno­globulin domain and mucin domain  3 (TIM3; also known as HAVCR2), CD160, 2B4 (also known as CD244) and B and T lymphocyte attenuator (BTLA) (FIG. 1). Importantly, although the interactions of these inhibitory receptors with their ligands are typically depicted between T cells and antigen-presenting cells (APCs), most of these interactions would not occur during initial T cell activation events. It is known that the majority of these inhibitory molecules are upregulated after the initial antigen-specific T cell receptor (TCR) interaction with dendritic cells (DCs). Furthermore, it is important to note that inhibitory molecules do not all necessarily have a role in exhaustion. Several studies have shown that the expression of these inhibitory receptors is associated with compromised function of antigen-specific T cells in patients with chronic infection or cancer 4–18. In one study19, tumour-specific T cells in melanoma-infiltrated lymph nodes were shown to express some of these markers — such as 2B4, PD1, TIM3 and LAG3 — although a subsequent study 20 showed that functional T cells isolated from patients with melanoma and from healthy donors can also express these inhibitory receptors. In fact, expression of these inhibitory receptors varies depending on T cell differentiation state (for example, some are expressed on naive T cells and on certain memory T cell populations) and on anatomical location (such as blood or lymph nodes)20. Thus, although the term ‘exhaustion

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REVIEWS marker’ is defined as molecules that are expressed on exhausted T cells, it should be kept in mind that such markers are not uniquely expressed on exhausted T cells but can also be associated with other differentiation states, including functional T cells. However, as antibodies that block these molecules can restore human T cell function in vitro and in animal models4–10,12,13,15–18, it is clear that these molecules represent potential novel therapeutic targets for chronic infection and cancer.

Dendritic cell

T cell

LAG3 CD4

Peptide– MHC class II

TCR

E-cadherin

KLRG1 BTLA

HVEM CD160 CD48

2B4

CD80

CTLA4

CD86

PD1 TIM3

PDL2 PDL1

HMGB1

Galectin 9 PtdSer

Dying tumour cell

Figure 1 | Receptors that negatively regulate T cell function.  Inhibitory receptors NatureT Reviews | Immunology and their ligands are depicted. The receptors include cytotoxic lymphocyte antigen 4 (CTLA4), programmed cell death protein 1 (PD1), lymphocyte activation gene 3 protein (LAG3), 2B4 (also known as CD244), killer cell lectin-like receptor subfamily G member 1 (KLRG1), CD160, B and T lymphocyte attenuator (BTLA) and T cell immunoglobulin domain and mucin domain 3 (TIM3). Their ligands include CD80, CD86, programmed cell death protein 1 ligand 1 (PDL1), PDL2, MHC class II, CD48, E‑cadherin, herpesvirus entry mediator (HVEM), galectin 9, phosphatidylserine (PtdSer) and high-mobility group box 1 protein (HMGB1). Only the interactions that mediate negative regulation are shown; some molecules have binding partners in addition to the ones shown that mediate other functions. In addition to the negative regulatory interactions, the interaction between the T cell receptor (TCR) and peptide–MHC class II is also shown as this is essential for T cell function. Note that dendritic cells also express MHC class I molecules and the negative regulatory interactions shown here can occur between dendritic cells and CD4+ T cells or CD8+ T cells. In this figure, PtdSer and HMGB1 are shown emanating from dying tumour cells. However, a stressed tumour cell could also upregulate these TIM3‑binding partners. Bidirectional negative signalling between PDL1 and CD80 has also been described; this interaction is detailed in FIG. 3.

Three molecules with inhibitory function have so far been clinically targeted: CTLA4, PD1 and LAG3. A therapeutic agent that blocks CTLA4, ipilimumab (Yervoy; Bristol-Myers Squibb), received US Food and Drug Administration (FDA) approval for use in the treatment of metastatic melanoma in 2011. CTLA4 is a marker that is upregulated following T cell activation. It clearly has an inhibitory role in T cell responses, as CTLA4‑deficient mice show a striking autoimmune-like phenotype. Additional studies have shown that CTLA4 has a role in both T cell and regulatory T (TReg) cell function21–26. However, these and other studies indicate that CTLA4 blockade does not function by reprogramming dysfunctional T cells. In addition, CTLA4 is not expressed on the surface of LCMV clone 13‑induced exhausted T  cells 9. The apparent disconnection between the upregulation of Ctla4 mRNA and the lack of cell surface CTLA4 protein may be related to CTLA4 being primarily found intracellularly due to continuous clathrindependent endocytosis27. In this Review, we focus on two inhibitory receptors that have recently been clinically targeted: PD1 and LAG3.

Targeting PD1 PD1 and T cell dysfunction. PD1 was cloned in 1992 as a type I transmembrane protein28; it has two known ligands — programmed cell death protein 1 ligand 1 (PDL1; also known as B7H1 and CD274)29,30 and PDL2 (also known as B7DC and CD273)31,32 — that are also type I transmembrane proteins. The expression patterns of PD1, PDL1 and PDL2 are complex 33. PD1 is generally expressed on T cells, natural killer (NK) cells, B cells and some myeloid cells. PDL1 is expressed at low levels on a wide range of non-haematopoietic cells and its expression can be upregulated in response to various stimuli. By contrast, immune cell types such as T cells, B cells and DCs constitutively express PDL1. PDL2 has a more restricted expression pattern compared with PDL1; cell types such as DCs and macrophages can inducibly express PDL2. T cells express PD1 only following activation when it functions to limit the effector phase of T cell differentiation33. PD1 has a key role in tolerance to self antigens and can also function as a ‘rheostat’ that modulates T cell responses34,35. Studies in chronic infection models have shown that blocking PD1 interactions can reverse T cell exhaustion4–7,17. Tumours can evade immune destruction by signalling through PD1; therefore, blocking PD1 signalling can induce an anti-tumour response36,37. A large number of studies show that PDL1 is expressed by a wide range of cancer cell types38. Thus, in the context of immune evasion by tumour cells, the prevailing view has been that tumour cells expressing PDL1 engage PD1 on tumour-infiltrating T cells and thereby inhibit T cell function (FIG. 2). Clinical trials targeting PD1. The therapeutic potential of targeting the PD1 pathway has led to the development of multiple clinical agents38. The treatment of cancer has so far been the primary focus for these agents, although they also have the potential to be used for the treatment of chronic viral infection39. In this section, we

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REVIEWS Myeloidderived cell Endothelial cell

IL-10 IL-6 and IL-10

–

DC

TReg cell

– PDL2

–

–

PD1

Hypoxic MDSC

–

?

– T cell APC

TFH cell

PDL1

–

Cell death

Conversion into pTReg cell

Tumour cell pTReg cell

–

–

B cell

NK cell

Figure 2 | Mechanisms of action of PD1, PDL1 and PDL2.  The potential roles of programmed cell death protein 1 (PD1), programmed cell death protein 1 ligand 1 (PDL1) and PDL2 in various cell types that can affect anti-tumour immunity are shown. The minus sign denotes interactions that negatively regulate T cell function and the question mark denotes an Nature Reviews | Immunology unknown effect. APC, antigen-presenting cell; DC, dendritic cell; IL, interleukin; pTReg cell, peripherally derived regulatory T cell; MDSC, myeloid-derived suppressor cell; NK, natural killer; TFH cell, T follicular helper cell.

limit our discussion to the published clinical trials for cancer 40–50 (TABLE 1). Overall, it is apparent that targeting the PD1 pathway has enormous potential to improve the outcomes of several cancers. Phase III clinical trials are ongoing and one agent, nivolumab (Opdivo; BristolMyers Squibb/Ono Pharmaceuticals), has recently been approved in Japan, and another, pembrolizumab (Keytruda; Merck & Co.), has been approved in the United States, for the treatment of metastatic melanoma. Several Phase I and Phase II clinical trials with these immune therapies have so far been completed and these trials provide several key findings. First some of these agents show high clinical response rates for metastatic melanoma41,43–46,50. Second, there is a lower overall incidence and severity of adverse events with these PD1–PDL1‑targeting agents compared with ipilimumab immune therapy 40,41,43–50. Third, there is evidence for synergy of these agents with other immune therapies such as ipilimumab42. Fourth, there is evidence of clinical efficacy in non-melanoma cancers including renal cell carcinoma, non-small cell lung cancer (NSCLC) and haematological cancers41,47–50. Finally, the clinical responses are often durable, not only in melanoma, but also in other cancer types.

Tumour expression of PDL1 as a biomarker of response? As mentioned above, the presumed mechanism of action for PD1 blockade in cancer is that it ‘releases the brakes’ on the anti-tumour T cell response at the tumour site; PD1- and PDL1‑specific antibodies are thought to prevent the interaction between PD1 on tumourinfiltrating T cells and PDL1 on tumour cells. If this is the case, tumour expression of PDL1 might predict clinical response to PD1 blockade. TABLE  2 lists the published clinical trials in which this has been examined40–43. Overall, these studies show some association between PDL1 expression by tumour cells and the clinical response to PD1 blockade. This positive association supports the presumed mechanism of action of PD1 blockade. However, it could also be related to the recent evidence that patients with PDL1‑positive tumours have better pre-existing antitumour immunity than patients with PDL1‑negative tumours. Several studies have evaluated the association between PDL1 expression in the tumour and disease prognosis (these studies were carried out in the general population of patients, not necessarily those who received PD1 blockade)38. Although early studies often found a negative association between PDL1 expression

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REVIEWS Table 1 | Published clinical trials targeting PD1 in patients with cancer Patient population

Dosing

Objective responses*

Correlative studies

Refs

Nivolumab (PD1‑specific monoclonal antibody)

‡

Metastatic melanoma, CRC, CRPC, NSCLC and RCC

Single dose (patients with 1/10 (10%) melanoma; 1/14 (7%) evidence of benefit after 3 months CRC; 0/8  CRPC; 0/6 NSCLC; and were eligible for repeat dosing) 1/1 (100%)  RCC

PDL1 expression in tumour was evaluated (see TABLE 2)

40

Advanced melanoma, NSCLC, CRPC, RCC and CRC (n = 296)

Multiple doses

26/94 (28%) melanoma; 14/76 (18%) NSCLC; 9/33 (27%) RCC; 0/17 CRPC; and 0/19 CRC

PDL1 expression in tumour was evaluated (see TABLE 2)

41§

Advanced melanoma

Multiple doses of ipilimumab and multiple doses of nivolumab given either in sequence or concurrently

7/33 (20%) in sequenced group and 21/53 (40%) in concurrent group

PDL1 expression in tumour was evaluated (see TABLE 2)

42

Advanced melanoma (n = 90)

Multiple doses of nivolumab with or without melanoma multipeptide vaccine

25% across all cohorts

PDL1 expression in tumour was evaluated (see TABLE 2)

43

Advanced melanoma (n = 107)

Multiple doses

31% across all cohorts

−

44§

Pembrolizumab (PD1‑specific monoclonal antibody)‡ Advanced melanoma

Multiple doses

44/117 (38%) across all cohorts

Regressing lesions showed dense CD8+ T cell infiltration

45§

Advanced melanoma (n = 173)

Multiple doses

26%

−

46§

Advance haematological Single dose malignancies

1/17 (5%) across all cohorts

−

47

DLBCL and PMBCL (post-AHSCT)

Multiple doses

18/35 (51%) of patients with measurable disease at screening post-AHSCT and before first dose of pidilizumab

Treatment increased the number of effector and memory T cells and increased the expression of IL‑7Rα on memory T cells

48

Relapsed follicular lymphoma

Multiple doses with rituximab

19/29 (66%)

Higher expression of T cell activation gene signature at baseline was associated with increased PFS, and increased PDL1 upregulation on peripheral blood T cells and monocytes at baseline was associated with response to therapy

49

9/52 (17%) melanoma; 5/49 (10%) NSCLC; 2/17 (12%) RCC; 1/17 (6%) ovarian cancer; 0/18 CRC; 0/14 pancreatic cancer; efficacy analysis not carried out for breast and gastric cancer

−

50

Pidilizumab (PD1‑specific monoclonal antibody)‡

BMS‑936559 (PD1‑specific monoclonal antibody)‡ Melanoma, NSCLC, CRC, RCC and ovarian, pancreatic, gastric and breast cancers (n = 207)

Multiple doses

*Objective responses being the sum of complete responses and partial responses. ‡Nivolumab is a fully human IgG4 monoclonal antibody; pembrolizumab is a humanized IgG4 antibody; pidilizumab is a humanized IgG1 antibody; BMS‑936559 is a fully human IgG4 antibody. Note, IgG4 antibodies do not mediate antibody-dependent cell-mediated cytotoxicity. §Cited references report on a single clinical trial. AHSCT, autologous haematopoietic stem cell transplant; CRC, colorectal cancer; CRPC, castrate-resistant prostate cancer; DLBCL, diffuse large B cell lymphoma; HCV, hepatitis C virus; IL‑7Rα, interleukin‑7 receptor α‑chain; NSCLC, non-small cell lung cancer; PD1, programmed cell death protein 1; PDL1, programmed cell death 1 ligand 1; PFS, progression-free survival; PMBCL, primary mediastinal large B cell lymphoma; RCC, renal cell cancer.

Merkel cell A cell type in the epithelium that is essential for the fine resolution of sensory stimuli; this cell type is malignantly transformed in Merkel cell carcinoma.

in the tumour and disease prognosis, it is now evident that this is not always the case. In recent studies, PDL1 expression by tumours is associated with the presence of tumour-infiltrating lymphocytes (TILs) and better prognosis. In NSCLC, the positive prognostic value of PDL1 expression was independent of age, stage and histotype51. A positive prognostic value of PDL1 expression was also shown in metastatic melanoma and Merkel cell carcinoma52,53. Interestingly, in patients with Merkel cell carcinoma, PDL1-positive tumours, TILs and patient prognosis were also positively associated with the

presence of Merkel cell polyomavirus (MCPyV) DNA in tumours, which strongly suggests that an endogenous T cell response against MCPyV was responsible for the anti-tumour response53. Thus, it is now appreciated that PDL1 expression by tumour cells is an indicator of a robust anti-tumour response — infiltration of the tumour microenvironment by activated T cells producing pro-inflammatory cytokines such as interferon‑γ (IFNγ) induces the upregulation of PDL1 on tumour cells54. This feedback loop is thought to be a mechanism of adaptive immune resistance by the tumour and also

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REVIEWS Table 2 | Objective responses in patients with PDL1‑positive versus PDL1‑negative tumours* Agent

OR in patients with PDL1‑positive tumours (%)‡

OR in patients with PDL1‑negative tumours (%)

Antibody clone

Refs

Nivolumab single agent

3/4 (75%)

0/5 (0%)

5H1

40,41

Nivolumab single agent

9/25 (36%)

0/17 (0%)

5H1

41

Ipilimumab and nivolumab (concurrent or sequential)

• Concurrent 6/13 (46%) • Sequential 4/8 (50%)

• Concurrent 9/22 (41%) • Sequential 1/13 (8%)

28‑8

42

Nivolumab with or without peptide vaccine

8/12 (67%)

6/32 (19%)

28‑8

43

*Data from published clinical trials. ‡PDL1‑positive tumours were defined as ≥ or >5% (depending on the study) of tumour cells that were positive for membranous staining with a PDL1‑specific antibody. OR, objective response; PDL1, programmed cell death protein 1 ligand 1.

involves the induction of other immunosuppressive factors by CD8+ TILs. The fact that these factors can be considered markers for pre-existing anti-tumour immunity provides an alternative explanation for the observed association between tumour cell PDL1 expression and response to therapy. Despite the evidence for an association between clinical response to PD1 pathway blockade and PDL1 expression by tumour cells, it is clear that not all patients with PDL1‑positive tumours respond to PD1 blockade and, conversely, some patients with PDL1‑negative tumours do respond (TABLE 2). Of note, the technical aspects of the immunohistochemistry assays for detecting PDL1 expression are still under development. Some methods and antibodies used in these assays may be more robust (that is, more sensitive and more specific) than others. Technical considerations aside, tumour expression of PDL1 is unlikely to be an ‘absolute’ biomarker for the selection of patients for treatment with PD1 pathway blockade for several reasons, including the dynamic regulation of PDL1 expression and the contribution of other suppressive mechanisms independent of PD1 and PDL1. Furthermore, there are many other ways that the PD1 axis could potentially affect anti-tumour immunity in addition to PDL1‑expressing tumour cells signalling to PD1 on T cells (see below). Dynamic regulation of PDL1 expression. The use of PDL1 as a biomarker for clinical response to PD1 pathway blockade is limited by the dynamic regulation of PDL1 expression. IFNγ has been well-established to induce the upregulation of PDL1 by many cell types including tumour cells33. Other cytokines including interleukin‑2 (IL‑2), IL‑7, IL‑15, IL‑21 and type I IFNs have also been shown to upregulate PDL1 expression by various cell types55–57. Thus, PDL1 expression probably fluctuates over time in response to changes in the inflammatory microenvironment, which leads to a poor correlation between expression levels at any one time point and response to therapy. Inhibitory mechanisms independent of the PD1 pathway. There are many other mechanisms by which the antitumour T cell response is downregulated, which would be unaffected by PD1 pathway blockade; for example, anti-tumour T cells can express other inhibitory receptors (FIG. 1). In addition, the tumour microenvironment

contains immunosuppressive factors such as indoleamine 2,3‑dioxygenase, arginase, vascular endothelial growth factor and transforming growth factor-β58. In the clinical trials of PD1 pathway blockade that assessed PDL1 expression as a biomarker for an anti-tumour response (TABLE 2), the threshold for defining PDL1 positivity was fairly low (≥5% of tumour cells stained positive with any intensity). Therefore, in many of the patients classified as having PDL1‑positive tumours, it is possible that only a small proportion of the TILs would actually encounter PDL1. In these patients, other inhibitory mechanisms would be particularly dominant. Role of PDL1 on non-tumour cells. The PD1 blocking agents that function in ‘releasing the brakes’ on antitumour T cells might affect the PDL1–PD1 interaction between the tumour cell and the TIL. However, expression of PDL1 by non-tumour cells is also likely to be an important mediator of T cell dysfunction. T cells can be regulated by PDL1 expressed by a variety of cell types including myeloid-derived cells, TReg cells and endothelial cells (FIG. 2). In the LCMV clone 13 model of T  cell exhaustion, PDL1 expression by haematopoietic cells was shown to limit the clonal expansion and the effector function of T cells59. In humans, PDL1 is expressed by myeloid-derived DCs and monocytes33. Blocking PDL1 or PDL2 enhances DC‑induced T cell proliferation and production of pro-inflammatory cytokines55,60. PDL1‑expressing myeloid-derived cells can have an important role in inhibiting anti-tumour T cells; for example, some chemotherapeutic agents induce the upregulation of PDL1 on immunosuppressive myeloidderived cells. In mice given preparative chemotherapy followed by adoptive T cell therapy, the addition of PD1 blockade augmented T cell function and enhanced the anti-tumour effect 61. TReg cells can function to dampen the response of effector T cells. PDL1 is expressed by CD4+CD25hi TReg cells and PD1 blockade has been shown to relieve the suppression of effector T cells by TReg cells62 (FIG. 2). In addition, PDL1 expressed by APCs has a role in the induction of peripherally derived TReg (pTReg) cells and in the maintenance of pTReg cell suppressive function63. Therefore, PD1 blockade may also indirectly enhance effector T cell responses by inhibiting the generation and the function of pTReg cells (FIG. 2).

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REVIEWS Two-photon intravital microscopy Laser-scanning microscopy that uses near-infrared laser light for the excitation of conventional fluorophores or fluorescent proteins. Owing to the deep tissue penetration of light, the main advantage is the ability to visualize live and intact specimens.

IFNγ upregulates PDL1 (and PDL2) expression on endothelial cells and tumour necrosis factor (TNF) has been shown to work together with IFNγ to induce further upregulation64,65. PDL1+ endothelial cells have been shown to inhibit the effector function of CD8+ T cells64,65 (FIG. 2). It has been shown that modulation of the T cell response by PDL1 expressed by endothelial cells has a physiological role in preventing immunopathology in a mouse model of myocarditis66. Tumour-specific T cells might also be regulated in this manner although this has not yet been reported.

Dendritic cell

T cell PDL1

–

CD80

CD28

+

CD86

CTLA4

–

CD8 Peptide– MHC class I

TCR

PDL1

PDL2

?

+

PD1

–

CD80

–

?

+

– PD1

RGMB

Alveolar epithelial cell or interstitial macrophage

PDL1

Tumour cell

Figure 3 | The network of receptors and ligands potentially affected by PD1 Nature Reviews | Immunology blockade.  The receptor–ligand interactions between programmed cell death protein 1 (PD1) on T cells and its ligands (programmed cell death protein 1 ligand 1 (PDL1) and PDL2) on dendritic cells, as well as other molecular interactions that could potentially be affected by perturbation of the PD1 axis are shown. The addition sign denotes interactions that positively stimulate T cell function; the minus sign denotes interactions that negatively regulate T cell function. Some molecules have ligands that are thought to exist on the basis of functional data, although the ligands have yet to be identified; these are indicated by a question mark. The molecular interactions shown here can occur between dendritic cells and CD4+ T cells or CD8+ T cells. CTLA4, cytotoxic T lymphocyte antigen 4; RGMB, repulsive guidance molecule B; TCR, T cell receptor.

Role of PDL2. The effect of PD1‑specific antibodies on the interaction between PD1 and PDL2 may also affect the anti-tumour function of T cells. Although some studies have detected PDL2 expression in the tumour microenvironment, PDL2 does not seem to be a dominant mediator of T cell inhibition at the tumour site67. PDL2 is expressed by various types of APCs (for example, monocytes, macrophages and DCs) and can also inhibit T cell responses31 (FIG. 2). Interestingly, although blockade of PD1–PDL2 interactions may enhance antitumour T cell responses, it probably also has effects on other T cell responses. Recent work has identified an interaction between PDL2 on lung DCs and repulsive guidance molecule B (RGMB) expressed by lung interstitial macrophages and alveolar cells68 (FIG. 3). This interaction was shown to facilitate the induction of the initial robust T cell clonal expansion that precedes respiratory immune tolerance. This finding raises the possibility that PDL2 mediates the rare but severe pneumonitis that has been observed in clinical trials of PD1 blockade: the availability of PDL2 for binding to RGMB may increase as a result of PD1 blockade, leading to vigorous clonal expansion of lung-resident T cells and immune-mediated pathology. Motility paralysis. An additional mechanism whereby PD1 signalling can lead to T cell dysfunction was elucidated in a study of LCMV clone 13 infection69. Using two-photon intravital microscopy, it was observed that exhausted T cells have impaired motility in the splenic marginal zone and red pulp. Blockade of PD1 resulted in increased T cell motility, IFNγ production and viral clearance. Thus, PD1 mediates motility paralysis of virus-specific T cells. In the context of a tumour, PD1 blockade may increase the motility and the function of tumour-specific T cells in lymphoid organs and may also facilitate serial cytolysis of tumour cells. Role of CD4+ T cells. Although CD8+ T cells are crucial mediators of anti-tumour immunity, the contribution of CD4+ T cells has also been increasingly appreciated; for example, adoptive T cell transfer studies in humans have shown that CD4+ T cells have significant clinical effects70,71. PD1 expression has been shown in chronic infection models to correspond to dysfunctional virusspecific CD4+ T cells72.TILs often include CD4+ T cells, and various subsets of T helper cells can be found within TILs, including T follicular helper cells (TFH cells). TFH cells support germinal centre B cell responses and recently have also been shown to have a positive effect on anti-tumour immunity, as a TFH cell gene signature in immune infiltrates in tumours is a predictor of survival in patients with breast cancer 73. Interestingly, TFH cells in the TIL population were shown to express PD1 ( REF. 73). A positive association between PD1+ TFH cells and prognosis seems counter-intuitive; however, it could be explained if PD1 is upregulated due to TFH cell activation but is not signalling inhibiting (for example, if PD1+ TFH cells do not come into contact with PDL1+ cells in the tumour microenvironment). Alternatively, PD1 may positively regulate TFH cells73.

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REVIEWS PD1 signals in myeloid-derived cells. PD1 is also expressed by macrophages, DCs and monocytes following their activation33. Evidence suggests that PD1 signalling in myeloid-derived cells has an immunomodulatory function (FIG. 2). PD1‑deficient mice have increased resistance to Listeria monocytogenes infection, even in the absence of recombination-activating gene 1 (RAG1) 74. PD1 expression on monocytes has been shown to be upregulated by putative pathogen-associated molecular patterns and by inflammatory cytokines present in the circulation of individuals infected with HIV‑1 (REF. 75). Triggering of PD1 on activated monocytes by PDL1 expressed by other cells was shown to induce IL‑10 production by the monocytes, which inhibited the proliferation of CD4+ T cells and their production of IFNγ and IL‑2 (REF. 75). It is possible that, in the tumour microenvironment, the presence of pro-inflammatory cytokines that are similar to those in the circulation of individuals infected with HIV‑1 may also induce the upregulation of PD1 on APCs within the tumour, leading to IL‑10‑induced inhibition of the anti-tumour T cell response. Myeloid-derived suppressor cells (MDSCs) have a role in downregulating anti-tumour T cell responses. It has been shown in patients with chronic hepatitis C virus infection that PD1 on MDSCs induces the production of IL‑10 by these cells, which suppresses CD8+ T cell proliferation, as well as the production of IFNγ and cytotoxic mediators such as granzyme B76.

T follicular helper cells (TFH cells). CD4+ T helper cells that function in providing help for B cell responses, including the formation of germinal centres and differentiation of B cells into antibody-producing plasma cells.

Germinal centre Located in peripheral lymphoid tissues (for example, the spleen), these structures are sites of B cell proliferation and selection for clones that produce antigen-specific antibodies of higher affinity.

Recombination-activating gene (RAG). RAG1 and RAG2 are essential for the rearrangement process that generates diversity in T cell receptor and antibody loci. Mice that are deficient for either of these genes fail to produce B cells or T cells owing to a developmental block in the gene rearrangement that is necessary for antigen receptor expression.

Reverse signalling via PDL1. In addition to the effects of signalling through PD1 expressed by T cells, PDL1 has been shown to induce signalling in tumour cells — a process termed ‘reverse signalling’ (FIG. 2). Reverse signalling through PDL1 expressed by tumour cells renders them refractory to death induced by tumourspecific CTLs and by other agents, including the protein kinase inhibitor staurosporine77,78. Reverse signalling through PDL1 has also been reported in DCs, in which it results in inhibition of DC maturation and increased IL‑10 production79. In the tumour setting, hypoxic conditions can induce expression of PDL1 on MDSCs. PD1 blockade under hypoxic conditions downregulates IL‑6 and IL‑10 production by MDSCs, leading to enhanced T cell activation80. PD1 and NK cells. PD1 is expressed by a subset of NK cells during chronic infections such as with Mycobacterium tuberculosis and HIV‑1 (REFS 81, 82). PD1 signalling in NK cells seems to negatively regulate their function; for example, PD1 blockade in vitro results in increased IFNγ production by NK cells from individuals infected with M. tuberculosis 81. Studies carried out in vitro using the PD1-specific monoclonal antibody pidilizumab (CT-011; CureTech) have shown that blocking PD1 can augment the antitumour activity of both T cells and NK cells83. This was also shown in transplantable tumour models in vivo83. In a Phase I clinical trial of pidilizumab for patients with haematological malignancies, pidilizumab showed evidence of clinical activity 47. A subsequent study showed

that PD1 is expressed by peripheral blood NK cells and that PDL1 is expressed by tumour cells from patients with multiple myeloma but not from healthy donors84 (FIG. 2). Furthermore, in vitro work showed that pidilizumab enhances the activity of NK cells against auto­ logous multiple myeloma cells and could potentially induce the migration of NK cells towards the bone marrow (that is, the tumour site)84. Given that NK cells can be regulated by PD1, this might be an important mechanism of action of PD1 blockade, particularly in patients with cancer types in which NK cell-mediated cytotoxicity has a key role in anti-tumour immunity. PD1 and B cells. It is clear that B cell responses are regulated in some way by PD1; mice that are deficient in PD1 develop antibody-mediated autoimmune disease85,86. However, the mechanisms are incompletely understood. Evidence suggests that germinal centre B cells may induce the upregulation of PD1 expression on germinal centre-associated T cells87. This suggests that dysfunction of TFH cells in the germinal centre induced by PD1 signalling prevents over-exuberant antibody production (FIG. 2). PD1 expression has also been reported on B cells, in which it exerts a negative regulatory effect 88 (FIG. 2). PD1 pathway blockade of B cells in vitro has been shown to enhance B cell proliferation and cytokine production88. In addition, TReg cells may directly suppress B cells via PD1 expressed by the B cells89. Furthermore, in macaques infected with simian immunodeficiency virus (SIV), in which the observed depletion of activated memory B cells following SIV infection is associated with more rapid disease progression, it was shown that the PD1+ B cells were preferentially depleted90. PD1 blockade ameliorated B cell depletion and increased the levels of SIV-specific antibodies. The current understanding is that T cells generally have a more crucial role in anti-tumour immunity than B cells. Therefore, the effect of PD1 blockade on B cell-mediated anti-tumour immune responses remains unknown. Other receptor–ligand interactions. The PD1–PDL1– PDL2 axis is clearly very complex, with expression of these surface molecules on many different cell types and the possibility for reverse signalling. Furthermore, other molecules impinge on this axis (FIG. 3). PDL1 interacts with CD80, which represents another checkpoint of T cell activity by inhibiting T cell responses91. Blocking PD1 may in turn make more PDL1 molecules available for increased binding to CD80, thereby augmenting T cell dysfunction. However, it is possible that the increased CD80 occupancy by PDL1 could decrease triggering of the negative regulatory molecule CTLA4 by CD80 and thus function as a checkpoint blockade of the CTLA4 pathway, although this possibility has yet to be examined. On the basis of functional data, PDL1 and PDL2 have also been shown to deliver a co‑stimulatory signal to T cells92–94; however, the identity of these putative receptors on T cells is unknown. Thus, several receptor–ligand

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REVIEWS networks could potentially be indirectly perturbed by PD1 pathway blockade and it is poorly understood how perturbation of one receptor–ligand interaction will affect other interactions.

Objective responses Reductions in tumour burden that satisfy the criteria for a complete or partial response.

Potential biomarkers of clinical response. There seems to be a positive association between tumour expression of PDL1 and the clinical response in the patient cohorts that have been assessed so far (TABLE 2). However, overall, the data indicate that tumour expression of PDL1 is not an ‘absolute’ biomarker of a clinical response. Other features of tumours have recently been assessed for predictive value95. In this study, using tissue that was previously assessed for an association between PDL1 expression and clinical response41, the presence of TILs was not an independent indicator of clinical response. PD1 expression by TILs showed a borderline correlation with clinical response and PDL1 expression by TILs did not correlate with objective responses (but did show a significant correlation with clinical benefit). PDL2 expression by tumour cells or TILs, CD4+/CD8+ T cell ratio, the presence of B cells and the presence of tumour necrosis did not show any association with clinical response. Correlative studies have indicated that T  cellmediated immunity is induced following PD1 pathway blockade. For example, increased CD8 + T cell infiltration into tumours and increased numbers of effector and memory T cells in the peripheral blood are observed following PD1 blockade45,48. Features of T cells may also be predictive biomarkers: in a combination study of rituximab and PD1 blockade with pidilizumab in patients with relapsed follicular lymphoma, improved clinical outcomes were associated with evidence of T cell activation in peripheral blood at baseline, higher expression of T cell activation gene signatures and higher expression of PDL1 on T cells and monocytes 49. These associations suggest that some degree of pre-existing anti-tumour immunity is required for a response to PD1 blockade and that PD1 blockade may not induce de novo activation of tumour‑specific T cells. Although the currently favoured explanation for the success of PD1–PDL1 blockade is thought to be associated with reversing exhaustion, other explanations — such as those that are described in this article — are possible. These alternatives should be considered as they may provide guidance for identifying biomarkers that predict patient response. Other possible biological activities of the PD1 axis that may contribute to response to therapy include: PDL1‑mediated T cell inhibition via expression by myeloid-derived cells, TReg cells or endothelial cells; PDL2‑mediated T cell inhibition by various types of APCs; the contribution of PD1‑expressing CD4+ T cells; the role of PD1 in the negative modulation of myeloid-derived cells; reverse signalling via PDL1 in tumour cells and other cells; negative regulation of NK cells via PD1; the role of PD1 in modulating B cell responses; and altered binding between other receptors and ligands that could be perturbed as a result of PD1 blockade.

The best predictor of clinical response is unlikely to be a single biomarker — it is more likely that it will be a combination of many biomarkers. Some may be directly related to the mechanism of action of PD1 blockade, others will perhaps be related to pre-existing anti-tumour immunity or to dominant immunosuppressive factors.

Targeting LAG3 LAG3 and T cell dysfunction. LAG3 was cloned in 1990 as a membrane protein96. The human LAG3 gene has 20% identity with the human CD4 gene and binds MHC class II molecules with high affinity. LAG3 is not expressed by resting T cells but is upregulated several days after T cell activation97. The known features and functions of LAG3 make it an appealing target for immune modulation, including its well-established role in the negative regulation of T cell function98. For example, blockade of LAG3 in vitro augments T cell proliferation and cytokine production99, and LAG3‑deficient mice have a defect in the downregulation of T cell responses induced by the superantigen staphylococcal enterotoxin B, by peptides or by Sendai virus infection100. LAG3 is upregulated on exhausted T cells compared with effector or memory T cells2. Although the establishment of chronic infection by LCMV clone 13 is not altered in LAG3‑deficient mice101, dual blockade of PD1 and LAG3 reverses T cell exhaustion and improves pathogen control in a synergistic manner in both LCMV clone 13 and Plasmodium falciparum infections9,101,102. In the context of cancer, LAG3 is upregulated on TILs10,19,103 and blockade of LAG3 can enhance anti-tumour T cell responses104. In addition, dual blockade of the PD1 pathway and LAG3 has been shown in mice and humans to be more effective for anti-tumour immunity than blocking either molecule alone10,105,106. Clinical trials targeting LAG3. The first clinical trials targeting LAG3 using antibody-mediated blockade in patients with cancer are underway — for example, a single agent trial for haematological malignancies (ClinicalTrials.gov, number: NCT02061761) and a trial testing single agent LAG3-specific antibody and LAG3 blockade in combination with PD1 blockade for solid tumours (ClinicalTrials.gov, number: NCT01968109). On the basis of the immunomodulatory role of LAG3 on T cell function in chronic infection and cancer, the predicted mechanism of action for LAG3‑specific mono­ clonal antibodies is to inhibit the negative regulation of tumour-specific effector T cells. Although LAG3 biology has not been as widely studied as that of PD1, there is evidence for pleiotropic roles of LAG3 that could invoke other mechanisms of action for LAG3 blockade. Other mechanisms of action of LAG3 blockade. LAG3 not only has a role in effector T cells but also in TReg cells107. LAG3 is expressed on activated TReg cells at higher levels than on effector T cells. LAG3 blockade has been shown to inhibit the suppressive activity of TReg cells in vitro and in vivo in a model of autoimmune pulmonary vasculitis107. Furthermore, it has been reported that TReg cells can acquire MHC class II

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REVIEWS Soluble LAG3 Lipid raft microdomain Dendritic cell

T cell

+

Membranebound LAG3

Peptide– MHC class II

–

Acquired by trogocytosis Peptide– MHC class II

Regulatory T cell

Figure 4 | Membrane-bound versus soluble LAG3.  The different outcomes of the Nature Reviews | Immunology interaction of lymphocyte activation gene 3 protein (LAG3) alternative spice variants with their MHC class II ligands are depicted. Signalling through membrane-bound LAG3 on T cells after it binds to MHC class II molecules negatively regulates T cell function. By contrast, signalling through MHC class II in lipid raft microdomains on a subset of dendritic cells after it is bound by soluble LAG3 (sLAG3) results in dendritic cell activation. In addition to interacting with MHC class II molecules on DCs, LAG3 is also reported to bind to MHC class II molecules that have been acquired by regulatory T cells in the process of trogocytosis. The addition sign denotes an interaction that positively stimulates cell function; the minus sign denotes an interaction that negatively regulates cell function.

Trogocytosis A process in which a cell can acquire portions of the cell membrane and molecules within the membrane from another cell. The term is generally used for immune cells.

Adjuvant An agent that improves an immune response, generally acting via stimulating antigen-presenting cells.

molecules via a process known as trogocytosis108. MHC class II‑expressing TReg cells could then mediate T cell suppression by engaging LAG3 expressed by effector T cells (FIG. 4). LAG3 expression has been observed on other immune cell types: the expression of LAG3 is induced on activated B cells in a T cell-dependent manner 109 and on approximately 20% of γδ T cells97. Although the function of LAG3 on these cell types is mostly unexplored, it has been noted that, in addition to a twofold increase in the number of T cells in aged LAG3‑deficient mice, these mice also have twice as many B cells, macrophages, granulocytes and DCs as their wild-type counterparts, which may suggest other roles for LAG3 in cellular homeostasis110. Although MHC class II molecules are generally expressed only by APCs, some cancer cells have been reported to express them as well. The LAG3 interaction with MHC class II molecules expressed by melanoma cells has been shown to protect the tumour cells from apoptosis111. Therefore, LAG3‑specific monoclonal antibodies could interfere with this protection from apoptosis, thus leading to enhanced tumour cell death. One of the main interests in LAG3 has been related to its role in inhibiting T  cell function. However, LAG3 also encodes an alternative splice variant that is translated to a soluble form of LAG3, which exhibits immune adjuvant activity.

Immune adjuvant activity of sLAG3. Although soluble LAG3 (sLAG3) is not the intended target of clinical trials using LAG3‑specific monoclonal antibodies, it may be informative to bear in mind the role of sLAG3 in T cellmediated immunity. Similarly to membrane-bound LAG3, the soluble form of LAG3 binds MHC class II. However, sLAG3 is thought to bind only to MHC class II molecules present in lipid raft microdomains on a minor subset of APCs112 (FIG. 4). Multiple studies show that sLAG3 functions as an immune adjuvant; for example, the immune adjuvant activity of a sLAG3–immunoglobulin fusion protein (sLAG3‑Ig) has been shown in mouse models to enhance anti-tumour T cell function in response to an irradiated tumour cell vaccine113. In in vitro experiments, the addition of sLAG3‑Ig during the activation of human CD8+ T cells that are specific for a range of tumour-associated antigens resulted in enhanced T cell clonal expansion114. There is evidence that sLAG3 has clinical importance in vivo, as overall survival is improved in patients with breast cancer who have higher levels of sLAG3 at the time of diagnosis compared with patients who have lower levels115. A sLAG3–Ig reagent (IMP321; Immutep) is currently in early phase clinical trials for use as an immune adjuvant. IMP321 consists of the four extracellular immunoglobulin-like domains of LAG3 fused to the Fc portion of human IgG1. In these trials, IMP321 was assessed in combination with antiviral vaccines or in combination with chemotherapy or other immune therapies for cancer 116–119. Although there is no known role for sLAG3 in directly inhibiting T cells, it is possible that sLAG3 affects the interaction of membrane LAG3 with MHC class II molecules and thus indirectly affects T cell function. Potential biomarkers of clinical response. The clinical efficacy of LAG3‑specific monoclonal antibodies remains to be seen. In order to explore biomarkers of potential clinical responses, expression of LAG3 on T cells within the tumour and in the blood should be evaluated. Given the potential effect of LAG3 on TReg cells, B cells, γδ T cells, macrophages and DCs, LAG3 expression on these cell types should also be evaluated. In light of the potential role of MHC class II molecules on TReg cells and tumour cells in mediating immune inhibition through LAG3, evaluating the expression of MHC class II on these cell types may also help to identify potential clinical responders. Finally, sLAG3 levels in the blood should also be measured in patients receiving LAG3‑specific monoclonal antibody. Although it is not clear if these two molecules would affect each other, any effect of sLAG3 levels on the clinical efficacy of LAG3 blockade (or vice versa) would shed light on the biology of this pathway.

Conclusion Agents targeting the PD1 pathway for cancer therapy have shown remarkable results in clinical trials, with one of the agents recently gaining FDA approval. Although one of the predicted mechanisms of action of PD1 blockade is to block PD1–PDL1 interactions at the tumour site, many studies show that PD1 pathway

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REVIEWS blockade could potentially invoke other mechanisms. A LAG3‑specific monoclonal antibody is in early phase clinical trials for cancer. LAG3 may also have multiple roles in immune regulation and the effect of LAG3 blockade in patients remains to be seen. As more agents targeting molecules that negatively regulate T cell function enter clinical trials, correlative studies that address the various roles of these molecules should be carried out. Defining the predominant mechanisms of action is needed in order to develop predictive biomarkers to aid in patient selection. This knowledge will also be instrumental in designing novel combination therapies involving blockade of other inhibitory receptors or stimulation of activating receptors.

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Note added in proof While this manuscript was in press, additional studies were published that evaluated the clinical effects of PD1 pathway blockade and investigated biomarkers that can predict the success of blocking this pathway. These studies include the use of PDL1‑specific monoclonal antibody MPDL3280A for treating various types of cancer, including bladder cancer, and a randomized Phase III trial of first-line nivolumab versus dacarbazine therapy for metastatic melanoma120–123. Biomarker evaluations in these studies support the association of pre-existing anti-tumour immunity with improved clinical response that was discussed in this article.

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Acknowledgements

The authors thank O. Chan for her contributions to this article.

Competing interests statement

The authors declare no competing interests.

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